Alzheimer's Disease (AD) is a chronic, neurodegenerative disorder characterized by a loss of cognitive ability and severe behavoral abnormalities in a patient leading to the eventual death of the patient. There are currently 2.5 to 4.0 million AD patients in the U.S. and 17 to 25 million worldwide. It is the fourth leading cause of death in Western cultures, preceded only by heart disease, cancer, and stroke. ARICEPT®, an acetylcholinesterase inhibitor has been approved by the FDA for decelerating the rate of decline of Alzheimer patients. However, it is effective only for a limited period of time and in some patients. Up to the present there is no definitive treatment or cure for this devastating disease.
Two microscopic deposits, i.e., neurofibrillary tangles (NFT) and senile amyloid plaques, were identified by Alois Alzheimer as the pathologic hallmarks of the disease. The neurofibrillary tangles consist of two 10 nm wide filaments twisted around each other, referred to as paired helical filaments (PHFs), a major component of which is phosphorylated tau. The phosphorylation of serine at amino acid 262 of the tau protein represents a crucial step leading to physiological dysfunction of tau. PHFs are intracellular and are found in many of the abnormal dendritic and axonal processes, or neurites that make up the periphery of senile amyloid plaques. The senile amyloid plaques consist of disorganized neurophil filaments in an area of up to 150 μm in cross section with an extra-cellular core of amyloid deposit. The cerebral amyloid plaques are ultrastructurally distinct from PHFs and consist of 4-8 nm wide filaments that are not wound together in pairs. The plaque core consists of aggregates of a peptide, initially referred to as A4, with a relative molecular mass (M) of about 4,000 (Masters et a1., Proc Natl Acad Sci USA, 1985, 82:4245-4249).
A partial amino acid sequence of A4, now renamed amyloid β peptide (or Aβ1-42), shows that it is similar to the amyloid β protein isolated from cerebral blood vessels of patients with Alzheimer's disease or Down's syndrome (Glenner and Wong, Biochem Biophys Res Comm, 1984; 120:885-890; 122:1131-1135).
Aβ1-42 has been hypothesized to be related to AD for a number of reasons. Firstly, in peripheral amyloidoses, e.g., primary light chain disease or secondary AA amyloidosis, large amyloid burdens strongly correlate with tissue and organ dysfunction. Secondly, amyloid plaque density positively correlates with premortem dementia scores in AD. Thirdly, Aβ1-42 deposition is the earliest neuropathological marker in AD and related disorders such as Down's syndrome, where it can precede NFT formation by 2-3 decades. Fourthly, β-amyloidosis is relatively specific to AD and related disorders. Fifthly, Aβ1-42 is toxic to neurons (Yankner et al., Science, 1990; 250:279-282). Lastly, missense mutations in the structural amyloid precursor protein (APP) gene cause early onset of familial AD (Goate et al., Nature, 1991; 349:704-706; Mullan et al., Nature Genetics, 1992; 1:345-347). Notably, one such mutation causes dramatic Aβ1-42 overproduction (Citron et al., Nature, 1992; 360:672-674).
In 1987, Kang et al. (Nature, 1987; 325:733-737) and three other groups (see 1987 status reports by Anderton, Nature, 1987; 325:658-659 and Barnes, Science, 1987; 235:846-847) independently cloned the gene from which Aβ1-42 is derived. This gene, now known as the amyloid precursor protein (APP), encodes a protein of 695 amino-acid residues with a MW of about 79,000 that is expressed in virtually all tissues. There are at least five splicing variants of APP, four of which contain the β-amyloid peptide sequence.
Four genes have been implicated in familial forms of AD. Three of the genes, βAPP, presenilin 1 and presenilin 2, when mutated, cause autosomal dominant early forms of AD. The fourth gene, Apolipoprotein E, has a naturally occurring polymorphic form, ApoE4, that represents a major genetic risk factor for the development of the disease. The concept that alterations in several distinct genes can lead to a chronic imbalance between Aβ1-42 production and its clearance, with the resulting aggregation of first the 42-residue and then the 40-residue peptide into cytotoxic plaques, is supported by available evidence. The evidence strongly suggests that defects in each of these four genes predispose the AD phenotype by (1) enhancing the production and/or the deposition of Aβ1-42 peptides or (2) by decreasing the clearance of ApoE4 from tissue (Selkoe, J Biol Chem, 1996; 271:18295-18298).
From available data, it appears that aggregated but not monomeric Aβ1-42 peptides can induce cell dysfunction and death in vitro by a range of presumably interrelated mechanisms. These include oxidative injury (Thomas et al., Nature, 1996; 380:168-171; Behl et al., Cell, 1994; 77:817-827), alterations in intracellular calcium homeostasis (Arispe et al., Proc Natl Acad Sci USA, 1993; 90:567-571), and cytoskeletal reorganization (Busciglio et al., Neuron, 1995; 14:879-888). Sufficient knowledge of some of the principal steps in the amyloid-induced cascade has emerged, even though the cascade hypothesis is hotly contested.
Pharmalogical approaches of identifying small molecules which could inhibit one or another step of the amyloid induced cascade is now well under way. Of particular interest are two approaches: attempts to interfere with the aggregation of Aβ1-42 peptides by decreasing the secretion of Aβ1-42 peptides from neuronal and glial cells or inhibit the toxicity that these extracellular aggregates produce on neurons and glial cells and their processes. A third approach attempts to control the specialized inflammatory response that appears to be triggered by aggregated Aβ1-42 (including microglial stimulation, activation of the classical complement cascade, cytokine release, and reactive astrocytosis) may prove to be of benefit to Alzheimer patients.
Aside from the above-mentioned pharmacological approaches for AD intervention, immunological interventions have also been attempted. Soloman et al. (Proc Natl Acad. Sci, 1996; 93:452-455; Proc Natl Aca. Sci, 1997; 94:4109-4112) showed that three specific monoclonal antibodies, directed toward a site in the N-terminal region of the human Aβ1-42 peptide, bind in varying degrees to preformed fibrils leading to their disaggregation and inhibition of their neurotoxic effect. The antibodies were also found to prevent the formation of fibrillar Aβ1-42. Solomon et al. (WO 01/18169) also attempted to prepare a phage display of an epitope of the Aβ1-42 peptide and administering the phage displayed epitope or peptide containing the epotope intraperitonially to mice to elicit antibodies to the Aβ1-42 peptide. In vitro testing with rat phenochromocytoma showed that a 1:5 dilution of the anitsera prevented the neurotoxicity of Aβ1-42. The antiserum at a dilution of 1:5 and 1:20 was also shown to disrupt the fibril structure of Aβ in vitro with extensive deterioration of fibril morphology. However, the adjuvant used was for the first injection was Complete Freund's Adjuvant with the incomplete Freund's Adjuvant for the second injection. The adjuvants used are entirely unsuitable for use in humans. Moreover, the levels of antibodies generated were too low to be effective despite the use of these harsh adjuvants.
Subsequently, Schenk et al. (Nature, 1999; 400:173-177) showed that immunization with Aβ1-42 peptide inhibits the formation of amyloid plaques and the associated dystrophic neurites in a mouse model of AD. However, due to the low immunogenicity of the Aβ1-42 peptide, the method employed required repeated administrations of the antigen with a harsh lesion-forming adjuvant to obtain the higher levels of anti-Aβ1-42 plaque antibodies necessary to affect plaque formation. Moreover, it was cautioned that immunization with Aβ1-42 might induce more accumulation of the toxic amyloid itself (Araujo, D M & Cotman, C W, Brain Res, 1992; 569, 141-145).
Despite these criticisms, additional studies in transgenic AD mice models through similar active immunization have lent credence to the immunoprophylaxis and immunotherapeutic approaches for AD. Janus et al. (Nature, 2000; 408:979-982) described Aβ1-42 peptide immunization in a mouse model for AD that reduced behavior impairment and plaques. Morgan et al. (Nature, 2000; 408:982-985) described Aβ1-42 peptide vaccination to prevent memory loss in the mouse model.
Direct support for the effectiveness of immune therapy came from the observation that peripheral administration of antibodies, monoclonal or polyclonal, against Aβ-peptide reduced amyloid burden (WO 99/27944; Bard et al., Nature Medicine, 2000; 6:916-919). Despite relatively modest serum levels, these passively administered antibodies, monoclonal 3D6 (anti-Aβ15) and 10D5 (anti-Aβ1-12) or polyclonal anti-Aβ1-42, were able to enter the central nervous system. There, the antibodies bound to plaques and induced clearance of pre-existing amyloid plaques. Bard et al., reported that when examined in an ex vivo assay with brain sections of PDAPP mice (i.e., mice transgenic for an APP mini-gene driven by a platelet-derived growth factor promoter) or AD patient brain tissue, antibodies against Aβ-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. This study demonstrated that passively administered antibodies against Aβ1-42 peptide and the Aβ1-42 N-terminus region reduced the extent of plaque deposition in a mouse model of AD; and that monoclonal antibodies or polyclonal antibodies elicited by site-directed vaccines are able to enter the CNS at therapeutically relevant levels.
Despite the promising findings of immunological intervention in mice model for AD, a vaccine against AD suitable for humans remains a long way off (Chapman, Nature, 2000; 408:915-916). The principal hurdles reside in the extensive work necessary to design and formulate an immunogenic composition that is uselful in humans before a practicable vaccine for AD can be achieved. Some of the issues that rely on experimental data for guidance are: (1) What is the specific target site for antibody recognition within the Aβ? (2) In what form should the immunogen be presented? (3) What other sites need to be included before an immunogen is achieved that will elicit a therapeutic level of antibody? (4) What is an effective vaccine delivery system employing a clinically acceptable adjuvant for humans?.
A major gap exists between what has been disclosed in the literature and what remains to be done. What is the suitable specific target site (i.e., the polymerized Aβ1-42 plaque or the monomeric soluble Aβ1-42 peptide) and how the specific site is to be engineered for immunological intervention. In spite of some 5,000 publications on Aβ1-42 over the past decade, the amyloid cascade hypothesis is hotly debated and the issue: the form in which Aβ1-42 should be used for intervention remains contentious. At the heart of the problem, argued by Terry and colleagues, is the weak correlation between fibrillar amyloid load and measures of neurological dysfunction (The Neuropathology of Alzheimer Disease and the Structure Basis of its Alterations, Ed. by Terry et al., Alzheimer Disease, p187-206, Lippincott Williams and Wilkins, 1999).
In AD patients, amyloid deposits often form at a distance from the site of neuron damage. The best correlation with pathological dementia is loss of synaptic terminals. However, the loss of synaptic terminals correlates poorly with amyloid load. If the manifestations of disease correlate weakly with amyloid load, then what is the role of Aβ? The article by Klein et al, titled “Targeting small Aβ1-42 oligomers: the solution to an Alzheimer's disease conumdrum?” (Trends in Neurosciences, 2001; 24:219-224) suggests that fibrils are not the only toxic form of Aβ, and perhaps not the neurotoxin that is most relevant to AD. Small oligomers and protofibrils, also termed as Aβ1-42 derived diffusible ligands (ADDLs), may also have potent toxic neurological activity.
An AD vaccine for successful immunological intervention will require an immunogen designed to elicit site-directed high affinity antibodies that bind to the senile plaques in the brain tissue to accelerate the clearance of the plaque by the Glial cells, and immunoneutralize the soluble Aβ-derived toxins.
The problem of raising high affinity site-directed antibodies against poorly immunogenic site-specific peptides have been known for decades. Immunologists and vaccinologists often resort to the classical hapten [peptide]-carrier protein conjugate approach as demonstrated in WO 99/27944. For the development of a site-directed vaccine against AD, Frenkel et al. attempted immunization against Aβ1-42 plaques through “EFRH”-phage administration (Proc Natl Acad. Sci 2000; 97:11455-11459, WO 01/18169) as mentioned above.
The approaches: using Aβ1-42 peptide aggregate or Aβ1-42 peptide fragment-carrier protein conjugates (WO99/27944) and using filamentous phage displaying “EFRH peptide” as the agents to induce immune responses against an amyloid deposit in a patient, are cumbersome and ineffective. For example, after the fourth immunization of 1011 phages displaying the EFRH epitope, >95% of the antibodies in the guinea pig immune sera are against the phages. Only a small population (<5%) of the antibodies is against the soluble Aβ1-42 peptide (Frenkel et al., Vaccine 2001,19:2615-2619, WO 01/18169).
Less cumbersome methods were described in EP 526,511 and WO 99/27944, which disclosed the administration of Aβ1-42 peptide to treat patients with pre-established AD and the administration of Aβ1-42 or other immunogens to a patient under conditions that generate a “beneficial” immune response in the AD patient. However, a review of WO99/27944 show that there are major deficiencies in the vaccine design disclosed therein.
In particular, the problem lies in the lack of a pharmaceutically acceptable and effective vaccine delivery system. WO99/27944 disclosed Aβ1-42 or active fragments of Aβ1-42 conjugated to a carrier molecule such as cholera toxin as the active vaccine component. See page 4 of WO 99/27944. Although page 5 taught that a pharmaceutical composition comprising the immunogen should be free of Complete Freund's Adjuvant [CFA], the only examples showing the efficacy of the Aβ1-42 vaccine for the treatment of AD in transgenic mice employed large doses of aggregated Aβ1-42 peptide in CFA. Despite repetitive recital of preferred adjuvants that are to be used with the disclosed immunogenic agents to enhance the immune response, experimental data showed that only the formulations employing CFA/ICFA provided a sufficiently high titer of anitbodies. See, page 25 of WO 99/27944. In example 1, the prophylactic efficacy of Aβ1-42 against AD was demonstrated in PDAPP mice. However, the formulations administered consist a dose of 100 ug per mouse of aggregated Aβ1-42 emulsified in Complete Freund's Adjuvant [CFA] (p34 of WO 99/27944) followed by multiple booster doses of the same Aβ1-42 peptide emulsified in Incomplete Freund's Adjuvant. In Example IX, the immune responses in mice to different adjuvants were studied. When the adjuvants: MPL, Alum, QS21, and CFA/ICFA were used with the purportedly potent immunogen AN1792 (i.e., aggregated human Aβ1-42), the level of antibodies to Aβ1-4 were reduced at a statistically significant level in comparison to mice that received the CFA/ICFA vaccines. See, Table 9, and pages 59-64 of WO 99/27944.
In the case where Aβ1-42 peptide fragments were used (human Aβ1-42 peptides of amino acids 1-5, 1-12, 13-28, and 33-42), each fragment was conjugated to sheep anti-mouse IgG as the protein carrier. In a later disclosure, the efficacy of antibodies to Aβ peptide fragments could only be shown by passive immunization with monoclonal antibodies (Bard et al., Nature Medicine 2000; 6:916-919). The efficacy of these fragments conjugated to sheep anti-mouse IgG was not shown. Therefore, the only immunogen shown to be effective was the aggregated Aβ1-42 peptide in CFA/ICFA.
Up to the present, all of the vaccine formulations shown to be effective employed CFA/IFA as the adjuvant. Peptide immunogens targeting Aβ1-42 have thus far been prepared by conjugation of the various Aβ1-42 fragments to sheep anti-mouse immunoglobulin, conjugation of synthetic Aβ13-28 via m-maleimidobenzoyl-N-hydroxysuccinimide ester to anti-CD3 antibody, or aggregated Aβ1-42 peptide alone. These immunogens, i.e., Aβ1-42 peptide alone or Aβ1-42 peptide-carrier protein conjugates, were emulsified with complete Freund's adjuvant for the first immunization, followed by subsequent boosts in incomplete Freund's adjuvant (Johnson-Wood et al., Proc Natl Acad Sci USA, 1997; 94:1550-1555; Seubert et al., Nature, 1992; 359:325-327; Schenk et al., Nature, 1999; 400: 173-177; Janus et al., Nature 2000; 408:979-982; and Morgan et al., Nature, 2000; 408:982-985). The formulations disclosed in WO 99/27944 or others using CFA and ICFA as adjuvants causes lesions and are too harsh for use in humans. Thus, none of the vaccine compositions for AD described in the prior art are suitable for use in humans.
In summary, despite statements suggesting the potential of Aβ1-42 peptide for the treatment of AD in view of the previous disclosures of Kline (EP 526,511), no problem solving vaccine formulations were really offered in WO99/27944 to address this key problem.
Another disadvantage with the peptide-carrier protein conjugates and Aβ1-42 aggregates is that these molecules are highly complex and are difficult to characterize and it is difficult to develop effective quality control procedures for the manufacturing process. A further disadvantage is that, Aβ1-42 peptide or its fragments are self molecules when administered to humans. Therefore, they are less immunogenic or non-immunogenic in humans. It is, thus, necessary to develop clinically acceptable vaccine formulations for administration in humans.
It is known that promiscuous Th epitopes may be employed to evoke efficient T cell help and may be combined with poorly immunogenic B cell epitopes to provide potent immunogens. Well-designed promiscuous Th/B cell epitopes chimeric peptides have been shown to be useful in eliciting Th responses and resultant antibody responses in most members of a genetically diverse population expressing diverse MHC haplotypes. Promiscuous Th from a number of pathogens, such as measles virus F protein and hepatitis B virus surface antigen, are known. Tables 1 and 2 lists many of the known promiscuous Th that have been shown to be effective in potentiating a short poorly immunogenic peptide, the decapeptide hormone LHRH (U.S. Pat. No. 5,759,551, and 6,025,468).
Potent Th epitopes range in size from approximately 15-40 amino acid residues in length, often share common structural features, and may contain specific landmark sequences. For example, a common feature of a Th is that it contains amphipathic helices, alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and with charged and polar residues dominating the surrounding faces (Cease et al., Proc Natl Acad Sci USA, 1987; 84: 4249-4253). Th epitopes frequently contain additional primary amino acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single Th epitope (Partidos et al., J Gen Virol, 1991; 72:1293). Most, if not all, of the promiscuous T cell epitopes fit at least one of the periodicities described above. These features may be incorporated into the designs of idealized artificial Th sites, including combinatorial Th epitopes. With respect to the design of combinatorial Th sites, lists of variable positions and preferred amino acids are available for MHC-binding motifs (Meister et al., Vaccine, 1995; 13:581-591). Furthermore, a method for producing combinatorial Th has been disclosed for combinatorial library peptides termed structured synthetic antigen library (Wang et al., WO 95/11998). Thus, the 1, 4, 5, 8 rule can be applied together with known combinatorial MHC-binding motifs to assign invariant and degenerate positions in a combinatorial Th site, and to select residues for the degenerate sites to vastly enlarge the range of immune responsiveness of an artificial Th. See, Table 2, WO 99/66957, and WO 95/11998.
Wang et al. (U.S. Pat. No. 5,759,551) suggested the use of immunostimulatory elements to render the self protein Amylin immunogenic. Wang et al. suggested the administration of immunogenic synthetic amylin peptides as vaccines for the treatment of non-insulin dependent diabetes mellitus (NIDDM), an amyloidogenic disease caused by overproduction of Amylin (column 19, lines 9-39, U.S. Pat. No. 5,759,551). Amylin is a 37 amino acid residues peptide hormone produced by the β cells in the islets of Langerhans. Overproduction of Amylin will result in the depositon of insoluble amyloid leading to amyloidogenic disease in the pancreas. Similar to the overproduction of Amylin, overproduction of the Aβ1-42 peptide will lead to the deposition of insoluble amyloid in the brain of AD patients. However, there is limited sequence homology between Amylin and the Aβ1-42 peptide. Only a short stretch of amino acids residues, VGSN, of Amylin32-35 corresponds to Aβ24-27. Antibodies produced against the Amylin peptide is not expected to be cross reactive to soluble Aβ1-42 peptides nor accelerate the clearance of amyloid plaques in the brain in view of the studies by Soloman et al. and Schenk et al., which showed that the sequence EFRH is critical.
It is the object of the invention to develop an immunogen that will enable the generation of high levels of high affinity antibodies against the N-terminal functional site of the Aβ1-42 peptide with high cross-reactivity to the senile plaques in the brain of AD patients. The anitbodies generated by binding to the Aβ1-42 peptide and the senile plaques is expected to accelerate the clearance of these plaques from the brain, promote fibril disaggregation, inhibit fibrillar aggregation, and immunoneutralization of the “soluble Aβ-derived toxins” [also termed as Aβ-derived diffusible ligands or ADDLs].
It is a further objective of the present invention to develop a vaccine delivery vehicle that is suitable for human or veterinary use for the prophylaxis and treatment of Alzheimer's Disease.